Specific Aims The emerging clinical success of immune checkpoint inhibitor cancer immunotherapy in the last four years has significantly extended survival of many types of human cancer patients. However, colorectal cancer (CRC), except for the small subset (4%) microsatellite instable (MSI) CRC, stands out as one of the few human cancers where anti-PD-1/PD-L1 immunotherapy has been unsuccessful. The mechanism under CRC non-response to anti-PD-1 is unknown. It is generally believed that the expression level of PD-L1 is a response predictor to anti-PD-1/PD-L1 immunotherapy. In the literature, the expression levels of PD-L1 in human CRC cells are controversial and it has been proposed that the lack of PD-L1 expression is the underlying mechanism of CRC non-response to anti-PD-1/PD-L1 immunotherapy. However, our preliminary data challenge this notion. Using a recently developed highly specific and FDA-approved anti-PD-L1 mAb, we have demonstrated that PD-L1 is abundantly expressed in tumor cells and myeloid-derived suppressor cells in human colon carcinoma. Furthermore, our preliminary data demonstrated that anti-PD-1 and anti-PD-L1 immunotherapy can effectively activate cytotoxic T lymphocytes (CTLs). In addition, we have shown that FasL of CTLs is essential for tumor growth control in an orthotopic colon cancer mouse model. Because CTLs suppress tumor growth through inducing tumor cell death, our observations suggest that CRC resistance to cell death is an underlying mechanism of CRC resistance to anti-PD-1/PD-L1 immunotherapy. Our central hypothesis is that CRC apoptosis resistance is an underlying mechanism of human CRC non- response to anti-PD-1 immunotherapy and ceramide analog IG7 is effective in potentiating CRC to FasL- induced apoptosis by anti-PD-1 antibody activated CTLs. We will test this hypothesis in this research project by pursuing two specific aims: 1) determine the efficacy of IG7 in promoting Fas clustering and FasL- induced apoptosis in colon carcinoma cells in vivo; and 2) determine whether IG7 increases the efficacy of anti-PD-1 immunotherapy to suppress human colon carcinoma in vivo. Successfully completion of the proposed studies has the potential to develop IG7 as a potentiating agent to overcome human colon cancer no response to anti-PD-1 immunotherapy.